Preface

Microorganisms are small, structurally simple, have a low evolutionary status, and are widely distributed. They have evolved into unique ecosystems in different living environments. The correlation analysis of the community structure characteristics of soil microorganisms has important practical significance for reducing crop planting costs and improving quality from the perspective of soil microecology. At the same time, the complex living environment has brought rich biological diversity, biochemical mechanisms, and metabolic pathways to soil microorganisms, making them have great potential in the production of new bioactive substances.

However, selecting suitable strains from the complex soil microbial community is always a challenging task. The general steps of traditional methods for screening bacterial strains are: bacterial strain collection → enrichment culture → purebred isolation → performance testing → bacterial strain preservation. This method is cumbersome and time-consuming, requiring a huge amount of work and repetitive labor. Therefore, in order to increase the screening efficiency, researchers have constantly constructed new screening theories and automated equipment. Many methods have also been successfully applied in the field of soil microbial screening, such as denaturing gradient gel electrophoresis, real-time fluorescent quantitative PCR and microarray.

The Tianmu Biotechnology High Throughput Microlite Droplet Culture omics System (MISS cell culture omics) is a miniaturized high-throughput single-cell culture and sorting equipment developed based on droplet microfluidics technology. It can separate and culture environmental microbial communities at the single-cell level, store the generated droplets in a highly permeable pipeline for incubation, and finally detect and sort them through optical signals (OD, fluorescence, chemiluminescence, etc.). The sorted droplets enter a porous plate for subsequent experiments.

Experimental Procedure

This case uses the Tianmu Biotechnology high-throughput micro upgrade droplet culture omics system as the basis to construct an efficient screening model for soil microorganisms.

The sample for this experiment is field soil. Weigh 2g of the sample, add 5 times the volume of physiological saline and glass beads, and shake overnight at 4 ℃. After settling, take the supernatant and count it on a hemocytometer. Dilute the supernatant into three concentration gradients (samples A, B, C) based on the bacterial content in the supernatant. Generate droplets on the machine and culture at room temperature. At the same time, samples D, E, and F were prepared at 5 times the concentration of the MISS cell, coated on plates, cultured at room temperature, and counted for colony count.

Cultivate in droplet trays for 15-45 days, select 5628 bacterial droplets from 60882 droplets, and coat a total of 88 plates at the same time. Count 761 bacterial colonies. The sequencing results showed that a total of 1264 bacteria were detected in the original soil, 86 bacteria were detected in the droplet system, and 73 bacteria were detected in the plate system (Figure 2). Among them, more than 50 strains of bacteria were identified in the droplets that could not be obtained in the plate, including 7 major categories of bacteria such as Brucella and defective short wave bacteria, which were only enriched in the droplets. Meanwhile, since the droplets are cultured after being wrapped in single cells, over 95% of the collected droplets can be identified and stored.

Conclusion

This case uses OD threshold as the screening basis to effectively isolate multiple microorganisms in the soil, and successfully detects 86 different microorganisms, which is 17.8% higher than traditional methods for screening types, while greatly reducing labor costs. This method demonstrates the effectiveness of microfluidic screening systems in soil microbial isolation.

Figure 1 Experimental process of soil microbial screening

Figure 2: Sequencing results of bacteria enriched in raw soil samples, droplets, and plates

Figure 3 Differences in bacterial species enriched in original soil samples, droplets, and plates